CN108642207A - A kind of detection method for quick and precisely identifying cowberry platymiscium - Google Patents
A kind of detection method for quick and precisely identifying cowberry platymiscium Download PDFInfo
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Abstract
The present invention proposes a kind of detection method for quick and precisely identifying cowberry platymiscium, is detected using following primer pair, the nucleotide sequence of the primer pair is as follows：The nucleotide sequence of VcSSR11 F and VcSSR11 R are respectively such as SEQ ID NO:1, shown in 2；The nucleotide sequence of VcSSR14 F and VcSSR14 R are respectively such as SEQ ID NO:3, shown in 4；The nucleotide sequence of VcSSR19 F and VcSSR19 R are respectively such as SEQ ID NO:5, shown in 6；The nucleotide sequence of VcSSR28 F and VcSSR28 R are respectively such as SEQ ID NO:7, shown in 8；The nucleotide sequence of VcSSR33 F and VcSSR33 R are respectively such as SEQ ID NO:9, shown in 10.In addition the present invention also proposes a kind of construction method of cowberry platymiscium allele collection of illustrative plates.Expand allele collection of illustrative plates with reference to this method, enriches available allele collection of illustrative plates quantity.
Description
Technical field
The present invention relates to molecular biology DNA marker technology and application fields, and in particular to one is pass through DNA marker skill
Art builds allele collection of illustrative plates quick and precisely to identify blueberry kind and its sibling species.
Background technology
Blueberry (blueberry) originates in North America, is that Ericaceae (Ericaceae) Vaccinium (Vaccinium) is planted
Object.Due to its fruit have very high nutritive value, from early in the twentieth century by domesticating and cultivating since, it is worldwide wide
General plantation.The introducing and planting of China's blueberry since nineteen eighty-three, as cultivation technique is continuously improved, blueberry industry development is swift and violent,
Form 4 main producing regions such as the Northeast, the Shandong Peninsula, the Yangtze river basin and Yunnan-Guizhou Plateau.
Blueberry is divided into cultigen and wild species, and according to the number of tree body size and chilling requirement, blueberry cultivation kind can divide again
For 5 northern high clump blueberry, southern high clump blueberry, half high clump blueberry, short clump blueberry and Vaccinium ashei types, each type includes again
A large amount of improved variety.The blueberry kind that the whole world has been reported at present has hundreds of, the blueberry kind almost all of the main cultivation of China
Quoted from foreign countries, and the kind utilized in China blueberry industry mainly has more than 10.The wild species of blueberry are widely distributed in me
State southwest and the Northeast, wild species are the important germ plasm resource of selection and breeding new blueberry.The correct identification of blueberry kind and parent
Edge relationship analysis is blueberry industry sound development, scientific development and the important prerequisite rationally utilized, but China's blueberry introduces a fine variety approach
More, kind information management is chaotic；It is simultaneous existing to there is the transliteration of kind name, free translation and certainly quasi- trade name in process of production
As causing seedling chaotic, the phenomenon that mixing the spurious with the genuine or adulterating often occur so that blueberry plants enterprise and farmer by weight
Big economic loss.
Traditional Variety identification is carried out based on blueberry leaf, flower, fruit and tree performance, and it is abundant that this requires assessor to have
Blueberry production experience and sturdy plant classification are gained knowledge, and the phenotypic character of blueberry is affected by planting environment, is led
It causes this discrimination method accuracy very low, differentiates that the situation of mistake usually occurs.Blueberry genomic DNA be not by growth place,
Landform, weather conditions and plant size are stabilized, because the main mode of reproduction of blueberry seedling is tissue cultures and cuttage,
The different single plants of same blueberry kind belong to clone, and genome sequence is completely the same, this is the discriminating of blueberry different cultivars
Provide material base.
Simple repeated sequence (Simple Sequence Repeat, SSR), also referred to as tandem repetitive sequence, are genomes
In a kind of codominance site for being widely present, have that polymorphism is high, reproducible, the advantages of being easy to detection, by using public
The EST of cloth, the genome sequence that sequencing is completed and transcript profile sequence can obtain the sites SSR.Although useful
In the SSR marker of the screening of blueberry kind and sibling species genetic map construction and functional gene, but suitable for cowberry platymiscium
EST-SSR wretched insufficiencies, by March 26th, 2018, the Vaccinium EST (EST) that GenBank is announced was only
22402, there is not yet the report that allele application of the graphic chart is identified in blueberry kind and its sibling species.As the second generation is surveyed
The continuous maturation of sequence technology, the procurement cost of high through-put sequence is lower and lower, and the sequencing period is shorter and shorter, this is from high-throughput sequence
The sites SSR are screened in column data to provide convenience.Therefore, filtered out from transcript profile sequence efficiently and accurately identification blueberry kind and
The sites SSR of its sibling species, can not only increase the quantity of Vaccinium EST-SSR molecular labelings, can also be blueberry kind and its
The quick and precisely identification of sibling species brings great convenience, and reference is provided for the selection and breeding of new blueberry.
Invention content
The present invention is the shortcomings that overcoming Vaccinium EST-SSR molecular labelings lazy weight in the prior art, to build Vaccinium
The allele collection of illustrative plates of plant provides a kind of method for quick and precisely identifying blueberry kind and its sibling species, increases Vaccinium point
While the quantity of son label, it is applied to identification and the Genetic relationship of the blueberry kind true and false, for new blueberry from now on
Selection and breeding provide reference.
It is achieved by the following technical solution：A kind of detection method for quick and precisely identifying cowberry platymiscium, uses
Following primer pair is detected, and the nucleotide sequence of the primer pair is as follows：
The nucleotide sequence of VcSSR11-F and VcSSR11-R is respectively such as SEQ ID NO:1, shown in 2；
The nucleotide sequence of VcSSR14-F and VcSSR14-R is respectively such as SEQ ID NO:3, shown in 4；
The nucleotide sequence of VcSSR19-F and VcSSR19-R is respectively such as SEQ ID NO:5, shown in 6；
The nucleotide sequence of VcSSR28-F and VcSSR28-R is respectively such as SEQ ID NO:7, shown in 8；
The nucleotide sequence of VcSSR33-F and VcSSR33-R is respectively such as SEQ ID NO:9, shown in 10.
Further, the cowberry platymiscium is from as follows：Berkeley, Lan Feng, Bridges tower, Ektar, Elliot,
Ze Xi, auspicious card, Anna, Biloxi, Lan Yu, emerald, Ao Ni'er, mist, flourishing age, Lai Gexi, pearl, seashore, Du Ke, Austria
Bundle gram indigo plant, Sharp's indigo plant, star, blue beauty, sapphire, your indigo plant, Hollister, McFarlin, Bei En, Robert Louis Stevenson, the Cangshan are got over
Tangerine, crow fruit, Jiangnan cowberry, Yunnan cowberry 1, Yunnan cowberry 2, Yunnan cowberry 3, without stalk cowberry 1, without stalk cowberry 2, blueberry 1
With blueberry 2.
In addition the present invention also proposes a kind of construction method of cowberry platymiscium allele collection of illustrative plates, includes the following steps：
Step 1：The extraction of Vaccinium DNA of plants；
Step 2：Simple repeated sequence Locus Analysis in Shoots in genome, includes the following steps：
Step 2.1PCR amplifications：
The DNA extracted using step 1 carries out PCR amplification as template, using primer pair described in claim 1；
Step 2.2 electrophoresis detection：
Above-mentioned amplified production electrophoresis is taken, result is photographed to record under ultraviolet lamp；
Step 2.3SSR Genotypings and Gene Mapper software statistics：
Above-mentioned PCR product and denaturant and internal reference mixing are chosen, 5min is denaturalized at 95 DEG C using PCR instrument, takes out immediately
It places on ice, is then placed in genetic analyzer and is analyzed, while fragment length is read using Gene Mapper softwares；
Step 3, analysis of genetic diversity and allele map construction
The allele length data that Gene Mapper are counted inputs Excel, according to unique allele length
Build its allele collection of illustrative plates.
Further, the cowberry platymiscium is from as follows：Berkeley, Lan Feng, Bridges tower, Ektar, Elliot,
Ze Xi, auspicious card, Anna, Biloxi, Lan Yu, emerald, Ao Ni'er, mist, flourishing age, Lai Gexi, pearl, seashore, Du Ke, Austria
Bundle gram indigo plant, Sharp's indigo plant, star, blue beauty, sapphire, your indigo plant, Hollister, McFarlin, Bei En, Robert Louis Stevenson, the Cangshan are got over
Tangerine, crow fruit, Jiangnan cowberry, Yunnan cowberry 1, Yunnan cowberry 2, Yunnan cowberry 3, without stalk cowberry 1, without stalk cowberry 2, blueberry 1
With blueberry 2.
Further, the step 2.1PCR, which is expanded, is specially：
Using the DNA that step 1 is extracted PCR amplification is carried out as template；
20 μ L reaction systems include：TaKaRa Premix rTaqTM10 μ L, 0.1 μ L of forward primer, 0.5 μ L of reverse primer,
0.4 μ L, the 10ng DNA profilings of M13 primers of fluorescent decoration, residual volume use distilled water polishing；
Response procedures：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C of extension 40s, 30 recycle
Afterwards, it is that 94 DEG C of denaturation 40s, 53 DEG C of annealing 40s, 72 DEG C of extension 40s carry out 8 cycles, last 72 DEG C of extensions to change cycling condition
8min。
Further, the M13 sequences of 18bp are uniformly added at the ends 5' of the forward primer：5’-
TGTAAAACGACGGCCAGT-3’。
Further, the M13 primers of the fluorescent decoration are that 5 ' ends use two kinds of fluorophors of Fam and Hex to modify respectively
M13 primers, wherein the sequence of M13 primers be 5 '-TGTAAAACGACGGCCAGT-3 '.
Further, the DNA extractions of the step 1 include the following steps：
Step 1.1 prepares DNA extracting solutions：2%CTAB, 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH 8.0；DNA
Dissolve buffer solution：10mM Tris；1mM EDTA；PH=8.0；
Vaccinium vegetable material is handled as follows in step 1.2：
1. the DNA extracting solutions and 8 μ L beta -mercaptoethanols of 400 μ L is added in 1.5mL centrifuge tubes, it is placed in 65 DEG C of water-baths
Preheat 15min；
2. taking the blueberry kind of 0.1g and its fresh blade of sibling species material, being put into a little polyethylene ratio in mortar coughs up alkane
Ketone K30 is added liquid nitrogen and is fully ground；Ground blade is transferred in the centrifuge tube of the extracting solution containing DNA, 65 are put into after mixing
Water-bath 30min in DEG C water-bath, shakes up once every 10min, mesophyll cell is made fully to crack；
3. after water-bath, taking out centrifuge tube and being cooled to room temperature；The volume ratio that 400 μ L precoolings are added is 24:1 chloroform/
Isoamyl mixed alkoxide solution centrifuges 15min after mixing well at 10 000rpm；
4. with the careful Aspirate supernatant of pipettor, be transferred in new 1.5mL centrifuge tubes, be added 400 μ L isopropanol and
The sodium acetate solution of the 3M of 40 μ L, gently overturn mixing, stand 15min to flocculent deposit is generated, then at 10 000rpm from
Heart 10min；
5. abandoning supernatant after centrifugation, bottom white precipitate is slightly carried to DNA and is carefully taken out, is transferred in 1.5mL centrifuge tubes, adds
The ethyl alcohol for entering 700 μ L 70% washes twice；Ethyl alcohol is abandoned after centrifugation, blots remaining ethyl alcohol with pipettor, white depositions are collected
On centrifuge tube side wall, it is put into 37 DEG C of air dry ovens and dries；
6. 500 μ L DNA dissolving buffer solutions are added to re-dissolve the DNA after drying, mixing is beaten using pipettor suction, is waited for
1 μ L RNase are added after being completely dissolved, is gently inhaled with pipettor and beats DNA solution, centrifuge tube is put into 37 DEG C of incubators after mixing
Middle heat preservation 1h, degrade extracting solution in RNA.
7. 4 μ L DNA solutions is taken to carry out electrophoresis on 1.5% Ago-Gel, its integrality is detected.Take half DNA molten
Liquid is diluted to 20ng μ L-1 and is reacted for subsequent PCR, and remainder is placed in -20 DEG C of preservations.
Possessed advantage is the present invention compared with the existing technology：
1. the present invention utilizes high clump blueberry ' Bridges tower ' kind chlorion stressed plants of this seminar early development
The sites SSR are found in blade transcript profile sequence, and 5 universal sites SSR with polymorphism are obtained through screening.
2. the present invention filters out 5 sites SSR has polymorphism in 38 blueberry kinds and its sibling species, can according to etc.
Position genome accurately judges blueberry kind or the resource true and false, is the new label being stabilized, can be directly by this
5 provided sites SSR of invention are applied on more blueberry kinds and its sibling species, expand allele figure with reference to this method
Spectrum enriches available allele collection of illustrative plates quantity.
3. 5 sites SSR of the present invention come from the blade of high clump blueberry kind ' Bridges tower ' chlorion stressed plants
Transcript profile is the sequence expressed simultaneously with functional gene or transcription factor, this is the function of studying these sites SSR and may have
It haves laid a good foundation.
4. quick and precisely identifying that blueberry kind and its method of sibling species, the present invention pass through to indigo plant the present invention provides a kind of
The amplification and genotyping in 5 simple repeated sequence (simple sequence repeat, SSR) sites in certain kind of berries genome,
Fast and accurately 38 parts of blueberry kinds and sibling species can be differentiated, parent can also be carried out to blueberry kind and its sibling species
Edge relationship analysis.The amplification in the sites SSR shares 5 primer pairs, can simultaneously be used in detection process.
5. the present invention establishes the allele collection of illustrative plates of 38 parts of blueberry kinds and its sibling species, which is not planted by plant
The influence of environment and cultivation step is blueberry kind and molecular identity card that sibling species are stabilized, can directly will be of the invention
The method established and 5 primer pairs provided are applied on more cowberry platymisciums, carry out germplasm identification, genetic diversity
Property analysis, genetic map construction and molecular mark.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is this hair
Some bright embodiments for those of ordinary skill in the art without having to pay creative labor, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is the pcr amplification product for marking VcSSR11, VcSSR28 and VcSSR33 in the Vaccinium vegetable material of part
Electrophoresis result.Wherein M be DL2000DNA marker (TaKaRa, Dalian), from up to down respectively length 2000,1000,
750, the standard DNA band of 500,250 and 100bp, a concentration of 50ng μ L-1.Sample 1 to 8 is respectively blueberry kind ' Bai Ke
Profit ', ' Lan Feng ', ' Ektar ', ' Biloxi ', ' Lan Yu ', ' emerald ', ' seashore ' and ' blue beauty ', 9 and 10 is respectively climing
More certain kind of berries kind ' McFarlin ' and ' Robert Louis Stevenson ', 11 and 12 be Vaccinium wild species Cangshan cowberry and crow fruit respectively.
Fig. 2 is blueberry kind and its sibling species allele collection of illustrative plates.Different Vaccinium plant genes are obtained by PCR amplification
Behind 5 sites SSR of group, Capillary Electrophoresis is carried out to its PCR product, according to fluorescence internal standard, is distinguished using Gene Mapper softwares
Equipotential gene size on 5 sites SSR is counted, is indicated with " digital 1/ number 2 ", such as on the sites VcSSR11, ' auspicious card ' etc.
Position genome is " 308/344 ", and Cangshan cowberry is " 316/316 ", and so on, construct 38 parts of Vaccinium vegetable materials
Unique allele collection of illustrative plates.
Fig. 3 is the affiliation clustering tree of different blueberry kinds and its sibling species.It is 5 groups that 38 parts of germplasm, which can gather,.Group I packets
9 blueberry kinds are included, wherein northern high clump blueberry and southern each 4 of high clump blueberry kind, while including that 1 Vaccinium ashei kind is (' blue
Jewel ').Group II is similar to group I situations, including northern high clump blueberry and southern each 4 of high clump blueberry kind；' pool west ', ' Lan Feng ' and
' affiliation of auspicious card ' 3 north high clump blueberry kind is closely.6 parts of germplasm gather in group III, wherein with 4 Cranberries
Include northern high clump blueberry kind (' Lan Yu ') and southern each 1 of high clump blueberry kind (' Lai Gexi ') in addition based on kind.In group IV
For 10 parts of Vaccinium wild species germplasm.Other 6 parts of germplasm gather in group V, (' blue beauty ' and ' expensive by 2 Vaccinium ashei kinds
It is blue '), 3 southern high clump blueberry kinds (' mist ', ' Ao Zhake is blue ' and ' Anna ') and 1 north high clump blueberry kind (' Ai Ke
Tower ') composition.
Specific implementation mode
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.
The present invention is further explained in the light of specific embodiments.
5 by the exploitation of high clump blueberry ' Bridges tower ' chlorion stressed plants blade transcript profile sequence in the present invention
The sites SSR and allele collection of illustrative plates are obtained by following methods：
(1) high clump blueberry ' Bridges tower ' chlorion stressed plants blade transcript profile sequence of this seminar early development is utilized
Row and MISA softwares find the sites SSR, and every SSR sequence generates 5 primers.Using software BatchPrimer3 design primers,
Primer screening condition is as follows：55-65 DEG C of primer length 18-28bp, Tm value, it is contemplated that amplified production length 100-500bp.It chooses at random
It selects and synthesizes 55 primers, ' Bridges tower ' is used to carry out PCR amplification verifications to 55 primers, choose that band is clear, big rostellum
The primer of conjunction is used as subsequent analysis.In the unified universal sequence (M13- for adding 18bp in 5 ' ends of designed forward primer
TGTAAAACGACGGCCAGT), (sequence is in addition synthesis 5' ends are modified using two kinds of fluorophors of Fam and Hex M13 primers
5’-TGTAAAACGACGGCCAGT-3’).Primer entrusts the synthesis of Shanghai Invitrogen trade Co., Ltd；
(2) using improvement CTAB (cetyltriethylammonium bromide, Hexadecy trimethyl ammonium
Bromide) the genomic DNA (the DNA extractions in i.e. follow-up specific practice) of method extraction blueberry kind and its sibling species material；
(3) PCR amplification is carried out by template of the genomic DNA of 38 parts of Vaccinium vegetable materials：In 20 μ L reaction systems
It is separately added into TaKaRa Premix TaqTM10 μ L, 0.1 μ L of forward primer, 0.5 μ L of reverse primer, the M13 primers of fluorescent decoration
0.4 μ L, 10ng DNA profilings.PCR amplification is carried out using Eppendorf Mastercycler, the specific steps are：94 DEG C of pre- changes
Property 3min；94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C of extension 40s, after 30 recycle, it is 94 DEG C of denaturation to change cycling condition
40s, 53 DEG C of annealing 40s, 72 DEG C of extension 40s, carries out 8 cycles, last 72 DEG C of extensions 8min.By 5 μ L PCR products and 1 μ L 6
After × Loading buffer mixings electrophoresis detection, ultraviolet lamp are carried out on 1.5% Ago-Gel containing 0.5 μ g/ μ L EB
Under photograph to record result.
(4) by the PCR product of about 100ng and 12 μ L denaturants and 0.25 μ L internal reference mixings, Eppendorf is used
Mastercycler PCR instruments are denaturalized 5min at 95 DEG C, and 5min on ice is placed in taking-up immediately, are then placed in 3130 heredity of ABI
Analyzer is analyzed, and corresponding allele size on 5 sites SSR is counted using Gene Mapper version 4.0,
The unique allele of Vaccinium different materials is analyzed using Microsoft Excel 2013.
Embodiment 1,
In the present invention, allele map construction method is as follows：
One, DNA is extracted
(1) DNA extracting solutions are prepared：2%CTAB, 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH8.0；DNA dissolves
Buffer solution：10mM Tris；1mM EDTA；PH=8.0；
(2) blueberry kind and its sibling species material are handled as follows：
1. the DNA extracting solutions and 8 μ L beta -mercaptoethanols of 400 μ L is added in 1.5mL centrifuge tubes, it is placed in 65 DEG C of water-baths
Preheat 15min；
2. taking the fresh blade of 0.1g blueberry kinds and its sibling species material, a little polyvinylpyrrolidone is put into mortar
K30 (PVP-K30) is added liquid nitrogen and is fully ground；Ground blade is transferred in the centrifuge tube of the extracting solution containing DNA, mixing
After be put into water-bath 30min in 65 DEG C of water-baths, shaken up once every 10min, mesophyll cell made fully to crack；
3. after water-bath, taking out centrifuge tube and being cooled to room temperature；The volume ratio that 400 μ L precoolings are added is 24:1 chloroform/
Isoamyl mixed alkoxide solution centrifuges 15min after mixing well at 10 000rpm；
4. with the careful Aspirate supernatant of pipettor, be transferred in new 1.5mL centrifuge tubes, be added 400 μ L isopropanol and
The sodium acetate solution of the 3M of 40 μ L, gently overturn mixing, stand 15min to flocculent deposit is generated, then at 10 000rpm from
Heart 10min；
5. abandoning supernatant after centrifugation, bottom white precipitate is slightly carried to DNA and is carefully taken out, is transferred in 1.5mL centrifuge tubes, adds
The ethyl alcohol for entering 700 μ L 70% washes twice；Ethyl alcohol is abandoned after centrifugation, blots remaining ethyl alcohol with pipettor, white depositions are collected
On centrifuge tube side wall, it is put into 37 DEG C of air dry ovens and dries；
6. 500 μ L DNA dissolving buffer solutions are added to re-dissolve the DNA after drying, mixing is beaten using pipettor suction, is waited for
1 μ L RNase are added after being completely dissolved, is gently inhaled with pipettor and beats DNA solution, centrifuge tube is put into 37 DEG C of incubators after mixing
Middle heat preservation 1h, degrade extracting solution in RNA.
7. 4 μ L DNA solutions is taken to carry out electrophoresis on 1.5% Ago-Gel, its integrality is detected.Take half DNA molten
Liquid is diluted to 20ng μ L-1It is reacted for subsequent PCR, remainder is placed in -20 DEG C of preservations.
Two, simple repeated sequence Locus Analysis in Shoots in genome
1, PCR amplification
(1) 20 μ L reaction systems include：
TaKaRa Premix TaqTM10 μ L, 0.1 μ L of forward primer, 0.5 μ L of reverse primer, the M13 primers of fluorescent decoration
0.4 μ L, 10ng DNA profiling (passing through the DNA for the Vaccinium vegetable material that above-mentioned DNA extraction method is extracted) and distilled water.
(2) response procedures：
94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C of extension 40s, after 30 recycle, change follows
Ring condition is that 94 DEG C of denaturation 40s, 53 DEG C of annealing 40s, 72 DEG C of extension 40s carry out 8 cycles, last 72 DEG C of extensions 8min.
2, electrophoresis detection：
5 μ L of above-mentioned amplified production are taken, 1 μ 6 × Loading of L buffer are added；In 1.5% containing 0.5 μ g/ μ L EB
Ago-Gel on electrophoresis, photograph to record result under ultraviolet lamp.
3, SSR Genotypings and Gene Mapper software statistics：
By the PCR product of about 100ng and 12 μ L denaturants and 0.25 μ L internal reference mixings, Eppendorf is used
Mastercycler PCR instruments are denaturalized 5min at 95 DEG C, and 5min on ice is placed in taking-up immediately, are then placed in 3130 heredity of ABI
Analyzer is analyzed, while reading fragment length using Gene Mapper softwares.
Three, allele map construction
The allele length data input Microsoft Excel 2013 that Gene Mapper are counted, according to
Unique allele length builds its allele collection of illustrative plates.
Test result：The present invention has found that 5 sites SSR show polymorphism on 38 different Vaccinium vegetable materials altogether,
Primer sequence is specifically shown in Table 1.
Table 1 is used to expand the primer sequence in the sites SSR in Vaccinium Plant Genome
Fig. 1 is that the PCR of the sites SSR VcSSR11, VcSSR28 and VcSSR33 in the Vaccinium vegetable material genome of part expand
Increase production object electrophoresis result.
With reference to figure 2, using VcSSR11, VcSSR14, VcSSR19, VcSSR28 and VcSSR33, this 5 sites SSR are built
The allele collection of illustrative plates of 38 parts of cowberry platymisciums, unique allele can effectively identify 38 parts of germplasm, including 21 Gao Cong
Blueberry kind, 3 Vaccinium ashei kinds, 10 Vaccinium wild species and 4 Cranberry kinds (Fig. 2).Current 38 parts of Vacciniums
Plant covers the blueberry kind of the main cultivation in China, if when current blueberry kind or its uncertain sibling species material, can use this
5 sites SSR in invention carry out PCR amplification to the genomic DNA of detected sample, and the allele of corresponding site is formed
The allele collection of illustrative plates corresponded in the present invention can determine the true and false of measuring samples.
Fig. 3 is the affiliation clustering tree of different blueberry kinds and its sibling species.It is 5 groups that 38 parts of germplasm, which can gather,.Group I packets
9 blueberry kinds are included, wherein northern high clump blueberry and southern each 4 of high clump blueberry kind, while including that 1 Vaccinium ashei kind is (' blue
Jewel ').Group II is similar to group I situations, including northern high clump blueberry and southern each 4 of high clump blueberry kind；' pool west ', ' Lan Feng ' and
' affiliation of auspicious card ' 3 north high clump blueberry kind is closely.6 parts of germplasm gather in group III, wherein with 4 Cranberries
Include northern high clump blueberry kind (' Lan Yu ') and southern each 1 of high clump blueberry kind (' Lai Gexi ') in addition based on kind.In group IV
For 10 parts of Vaccinium wild species germplasm.Other 6 parts of germplasm gather in group V, (' blue beauty ' and ' expensive by 2 Vaccinium ashei kinds
It is blue '), 3 southern high clump blueberry kinds (' mist ', ' Ao Zhake is blue ' and ' Anna ') and 1 north high clump blueberry kind (' Ai Ke
Tower ') composition.
Blueberry kind according to the present invention almost enumerates all main breeds of China, utilizes 5 sites SSR institute structures
The allele collection of illustrative plates of the blueberry kind and its sibling species built, which has, expands space, and in given known kind, can utilize 5
One or more new unique alleles that material is added of combination supplement in the sites SSR.If blueberry kind to be detected is not in this example
38 listed also assess the affiliation of kind to be detected and known kind using 5 sites SSR provided by the invention,
Reference is provided for cultivar identification.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these are changed or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Sequence table
<110>Zhejiang Normal University
<120>A kind of detection method for quick and precisely identifying cowberry platymiscium
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gctccctctt tcgcttcttt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aaaaatcagg ttcggttcca 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cagtttgcac atcacccttg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggatacat ggaccttgcc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ccaaccctcg tactcttcca 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cccacctcag aaaaccgata 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gcagcgaacc ctaaactcaa 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
acggagctgg ctcacactat 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ggtttgggtt tgcctctctc 20
<210> 10
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
acgccgtgat ctgcagtag 19
Claims (8)
1. a kind of detection method for quick and precisely identifying cowberry platymiscium, which is characterized in that it is detected using following primer pair,
The nucleotide sequence of the primer pair is as follows：
The nucleotide sequence of VcSSR11-F and VcSSR11-R is respectively such as SEQ ID NO:1, shown in 2；
The nucleotide sequence of VcSSR14-F and VcSSR14-R is respectively such as SEQ ID NO:3, shown in 4；
The nucleotide sequence of VcSSR19-F and VcSSR19-R is respectively such as SEQ ID NO:5, shown in 6；
The nucleotide sequence of VcSSR28-F and VcSSR28-R is respectively such as SEQ ID NO:7, shown in 8；
The nucleotide sequence of VcSSR33-F and VcSSR33-R is respectively such as SEQ ID NO:9, shown in 10.
2. detection method according to claim 1, which is characterized in that the cowberry platymiscium is from as follows：Berkeley, indigo plant
Rich, Bridges tower, Ektar, Elliot, Ze Xi, auspicious card, Anna, Biloxi, Lan Yu, emerald, Ao Ni'er, mist,
Flourishing age, Lai Gexi, pearl, seashore, Du Ke, Ao Zhake indigo plant, Sharp's indigo plant, star, blue beauty, sapphire, your indigo plant, Hollister,
McFarlin, Bei En, Robert Louis Stevenson, Cangshan cowberry, crow fruit, Jiangnan cowberry, Yunnan cowberry 1, Yunnan cowberry 2, Yunnan cowberry
3, without stalk cowberry 1, without stalk cowberry 2, blueberry 1 and blueberry 2.
3. the construction method of cowberry platymiscium allele collection of illustrative plates, which is characterized in that include the following steps：
Step 1：The extraction of Vaccinium DNA of plants；
Step 2：Simple repeated sequence Locus Analysis in Shoots in genome, includes the following steps：
Step 2.1PCR amplifications：
The DNA extracted using step 1 carries out PCR amplification as template, using primer pair described in claim 1；
Step 2.2 electrophoresis detection：
Above-mentioned amplified production electrophoresis is taken, result is photographed to record under ultraviolet lamp；
Step 2.3SSR Genotypings and Gene Mapper software statistics：
Above-mentioned PCR product and denaturant and internal reference mixing are chosen, is denaturalized 5min at 95 DEG C using PCR instrument, taking-up is placed immediately
On ice, it is then placed in genetic analyzer to be analyzed, while fragment length is read using Gene Mapper softwares；
Step 3, analysis of genetic diversity and allele map construction
The allele length data that Gene Mapper are counted inputs Excel, is built according to unique allele length
Its allele collection of illustrative plates.
4. construction method according to claim 3, which is characterized in that the cowberry platymiscium is from as follows：Berkeley, indigo plant
Rich, Bridges tower, Ektar, Elliot, Ze Xi, auspicious card, Anna, Biloxi, Lan Yu, emerald, Ao Ni'er, mist,
Flourishing age, Lai Gexi, pearl, seashore, Du Ke, Ao Zhake indigo plant, Sharp's indigo plant, star, blue beauty, sapphire, your indigo plant, Hollister,
McFarlin, Bei En, Robert Louis Stevenson, Cangshan cowberry, crow fruit, Jiangnan cowberry, Yunnan cowberry 1, Yunnan cowberry 2, Yunnan cowberry
3, without stalk cowberry 1, without stalk cowberry 2, blueberry 1 and blueberry 2.
5. construction method according to claim 4, which is characterized in that the step 2.1PCR, which is expanded, is specially：
Using the DNA that step 1 is extracted PCR amplification is carried out as template；
20 μ L reaction systems include：TaKaRa Premix rTaqTM10 μ L, 0.1 μ L of forward primer, 0.5 μ L of reverse primer, fluorescence
0.4 μ L, the 10ng DNA profilings of M13 primers of modification, residual volume use distilled water polishing；
Response procedures：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 40s, 54 DEG C of annealing 40s, 72 DEG C extend 40s and change after 30 recycle
Become cycling condition and carries out 8 cycles, last 72 DEG C of extensions 8min into 94 DEG C of denaturation 40s, 53 DEG C of annealing 40s, 72 DEG C of extension 40s.
6. construction method according to claim 5, which is characterized in that uniformly add 18bp's at the ends 5' of the forward primer
M13 sequences：5’-TGTAAAACGACGGCCAGT-3’.
7. construction method according to claim 6, which is characterized in that the M13 primers of the fluorescent decoration are that 5 ' ends are distinguished
The M13 primers modified using two kinds of fluorophors of Fam and Hex, the wherein sequence of M13 primers are 5 '-
TGTAAAACGACGGCCAGT-3’。
8. construction method according to claim 7, which is characterized in that the DNA extractions of the step 1 include the following steps：
Step 1.1 prepares DNA extracting solutions：2%CTAB, 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH 8.0；DNA dissolves
Buffer solution：10mM Tris；1mM EDTA；PH=8.0；
Vaccinium vegetable material is handled as follows in step 1.2：
1. the DNA extracting solutions and 8 μ L beta -mercaptoethanols of 400 μ L is added in 1.5mL centrifuge tubes, it is placed in 65 DEG C of water-baths and preheats
15min；
2. taking the blueberry kind of 0.1g and its fresh blade of sibling species material, a little polyvinylpyrrolidone is put into mortar
K30 is added liquid nitrogen and is fully ground；Ground blade is transferred in the centrifuge tube of the extracting solution containing DNA, 65 DEG C are put into after mixing
Water-bath 30min in water-bath shakes up once every 10min, mesophyll cell is made fully to crack；
3. after water-bath, taking out centrifuge tube and being cooled to room temperature；The volume ratio that 400 μ L precoolings are added is 24:1 chloroform/isoamyl
Mixed alkoxide solution centrifuges 15min after mixing well at 10 000rpm；
4. with the careful Aspirate supernatant of pipettor, it is transferred in new 1.5mL centrifuge tubes, the isopropanol and 40 μ L of 400 μ L is added
3M sodium acetate solution, gently overturn mixing, stand 15min to flocculent deposit is generated, then centrifuged at 10 000rpm
10min；
5. abandoning supernatant after centrifugation, bottom white precipitate is slightly carried to DNA and is carefully taken out, is transferred in 1.5mL centrifuge tubes, is added
The ethyl alcohol of 700 μ L 70% washes twice；Ethyl alcohol is abandoned after centrifugation, remaining ethyl alcohol is blotted with pipettor, and white depositions are collected
On centrifuge tube side wall, it is put into 37 DEG C of air dry ovens and dries；
6. 500 μ L DNA dissolving buffer solutions are added to re-dissolve the DNA after drying, mixing is beaten using pipettor suction, is waited for completely
1 μ L RNase are added after dissolving, is gently inhaled with pipettor and beats DNA solution, centrifuge tube is put into 37 DEG C of incubators after mixing and is protected
Warm 1h, degrade extracting solution in RNA；
7. 4 μ L DNA solutions is taken to carry out electrophoresis on 1.5% Ago-Gel, its integrality is detected；Half DNA solution is taken,
It is diluted to 20ng μ L-1It is reacted for subsequent PCR, remainder is placed in -20 DEG C of preservations.
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| CN114292901A (en) * | 2021-09-08 | 2022-04-08 | 安徽师范大学 | Method for screening real-time fluorescent quantitative PCR (polymerase chain reaction) reference genes of vaccinium bracteatum |
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